Study On The Mechanism Of The Effect Of Redox Substances On The Formation And Development Of Fruiting Body In Hypsizygus Marmoreus

Fruiting body formation is a complex developmental problem,involving a series of metabolic processes,such as genetics,physiology and biochemistry and so on.It was found that addition of redox substances saponin,hydrogen peroxide and hydrogen-rich water(HRW)in a certain concentration range could promote fruiting body formation in Hypsizygus marmoreus.Previous studies have shown that redox active substances can regulate the level of reactive oxygen species(ROS)in cells.This research aims to start with the effects of redox active substances on phenotypic of fruiting bodies,ROS levels and related enzyme activities to study the changes of redox active substances on the expression of genes related to the regulation of ROS levels.This research aims to investigate the effects of redox active substances on the phenotypes of fruiting bodies,ROS levels,enzyme activities and gene expression related to the regulation of ROS levels in H.marmoreus.This research will expound the function and action of key genes,including the genes of glutathione peroxidase,regulating intracellular ROS levels by gene silencing.And this study will analyze the ROS levels,physiological and biochemical,fruiting body formation and other genes expression in silent transformants.The correlation mechanism of adding saponins,hydrogen peroxide and hydrogen-rich water on the formation and development of fruiting body in Hypsizygus marmoreus was discussed.The following results were obtained:In the industrial production of H.marmoreus,adding a certain concentration of saponins could promote the average yield of the strains with 60 days' culture time and 85 days' culture time to be 196.0 g/bottle and 208.9 g/bottle,respectively.The average yield of the control group was 189.0 g/bottle.Then,we measured the contents of intracellular malondialdehyde(MDA)and hydrogen peroxide(H2O2)and the activity of superoxide dismutase(SOD),catalase(CAT),glutathione reductase(GR),and glutathione peroxidase(GPX)in four developmental stages treated with 0.001%saponins.The results showed that the addition of saponins alleviated the mechanical damage caused by the process of scratching and enhanced the anti-oxidation ability of the cells.And then the relative expression of the key genes of saponins synthesis(farnesyl pyrophosphate synthase FPS,squalene synthase SS and squalene epoxidase SE),the genes of antioxidant enzyme(SOD,CAT,GR and GPX)and signal genes PKA and RAS in four growth stages of H.marmoreus treated with saponins was studied.Finally,the gene expression and metabolite level in mycelium and fruiting body of H.marmoreus were studied by transcripts and metabonomics techniques.The contents of amino acids and their intermediates in mycelial metabolites treated with saponins increased significantly,especially the content of 6-trehalose,a signal regulator,increased by 3.49 times.The data of the transcriptional group showed that the difference of mycelia regeneration stage between the saponins treatment group and the control group was the largest,and the number of differentially expressed genes reached 1380,among which a large number of functional genes related to cell metabolism synthesis were significantly up-regulated.Particularly,there were significant differences in gene expression between small GTPase-mediated and ras protein signal transduction.The results indicated that the addition of saponins in H.marmoreus could alleviate the mechanical damage and promote the recovery of hyphae,and multiple signaling pathways such as trehalose 6-phosphate,regulation of small GTPase mediated signal transductionand regulation of Ras protein signal transduction may be related to the development of fruiting bodies.The addition of suitable concentration of hydrogen peroxide after scratching can promote the formation of fruiting body of H.marmoreus,which is faster than the control group in the development process.And the treatment of hydrogen peroxide with 6mM concentration had the best effect on increasing yield,whose average yield increased by 7.92%.We measured the content of hydrogen peroxide and the activity of anti-superoxide anion,superoxide dismutase(SOD)and catalase(CAT)during the developmental stages of H.marmoreus treated with hydrogen peroxide.And the mycelium treated with H2O2 was studied by transcript and proteome technique,and the expressed protein was verified by mass spectrometry-targeted proteome quantitative technique(PRM).Finally,the effect of H2O2 treatment on the fruiting body development of H,marmoreus was analyzed by transcript group technique.There were significant differences between addition hydrogen peroxide of 24h and the control group,which involved a large number of biological regulation,metabolic process,signaling and other functional genes.And further metabolic pathway analysis showed that the catalytic activity and hydrolase activity-related pathways enriched more genes.The above results indicated that the addition of H2O2 to H.marmoreus could change the intracellular redox level and stimulate the biological regulation,metabolic process and signal expression of the cells under oxidative stress.And the expression of genes such as biological regulation,metabolic process,signal,and so on,could be stimulated by oxidative stress.The metabolic pathways of catalytic activity and hydrolase were initiated to promote the fruiting body development of H.marmoreus.The addition of hydrogen-rich water(HRW)with selective antioxidant activity after scratching can promote the formation of fruiting body of H.marmoreus,which is faster than the control group in the development process.The addition of HRW can increase the average yield by 11.76%and 21.30%.The physiological function and molecular mechanism of hydrogen-rich water in relieving three different types of stress in mycelium of H.marmoreus were further studied.The results showed that certain concentration of hydrogen-rich water treatment significantly reduced the toxicity of CdCl2,NaCl and H2O2 to mycelium,leading to significantly improve the growth rate and biomass.It was significantly decreased the formation of malondialdehyde(MDA)in mycelium cells.Meanwhile hydrogen-rich water treatment significantly increased the activities of antioxidants(SOD,CAT and GR)as well as the gene expressions of these antioxidants(SOD,CAT and GR)at the mRNA level.In vivo detection of reactive oxygen species(ROS),including H2O2 and O2-,as well as lipid peroxidation provided further evidence that HRW could significantly improve tolerances of CdCl2,NaCl and H2O2.In addition,pyruvate kinase and its gene expression were activated in the mycelium treated with hydrogen-rich water,suggesting that hydrogen-rich water treatment could promote glucose metabolism.Finally,the effects of hydrogen-rich water treatment on mycelium cells were analyzed by transcript set technique.The results showed that phosphorus,nitrogen and ion transmembrane transport genes were enriched.Besides,the genes expression of glutathione-disulfide reductase,glutathione disulfide bond oxidoreductase and peptide disulfide bond oxidoreductase were enhanced.In conclusion,adding of hydrogen-rich water to H.marmoreus can alleviate the stress damage caused by the external environment of the mycelium stage and promote the recovery of the hyphae after scratching.Therefore,it is accelerated the fruiting body formation of H.marmoreus.Furthmore,it is studied the function of glutathione peroxidase gene(gpx)by using the established silencing transformation system in the fruiting body development of H.marmoreus.The silencing efficiency of two gpx-silencing transformants,gpxil2 and gpxi19,were 75%and 88%,respectively.The silencing transformants were fruiting-tested,and whose growth and development of fruiting bodies were observed by adding hydrogen peroxide,and the content of oxidoreductase and the activity of oxidoreductase were measured.At last,the activity of lignocellulose degrading enzyme in cell and the enzyme activity of mitochondrial complex in the mitochondria were measured.The morphological changes of the transformant mitochondria were observed by transmission electron microscopy(TEM).The above results showed that glutathione peroxidase gene silencing strain showed lag in the fruiting body formation,and the activities of lignin peroxidase,laccase and cellulase related to substrate degradation decreased.The activity of intracellular mitochondrial complex(?)was up-regulated significantly.And the mitochondrial morphology of the gpx-silenced strain was abnormal by transmission electron microscopy(TEM).In conclusion,it is firstly studied systematically the effects of different redox substances on the formation and development fruiting bodies of H.marmoreus.The differential expression data of transcription,proteome and metabolism in the development stages of fruiting body under different redox treatments were obtained,which laid a data foundation for the further study of gene function in the development of fruiting body of H.marmoreus.The function of glutathione peroxidase(GPX)gene in the development fruiting body of H.marmoreus was studied,which was found that was very important to maintain the fruiting body development process of H.marmoreus.